Essentially all living systems produce cell surface structures to rigidify cells, form protective coats, or facilitate cell adhesion and migration. Microbial ?cell walls? usually perform protective functions for survival under detrimental conditions, to reduce the efficacy of their host?s innate immune response, or to form 3-dimensional meshworks, called biofilms. Common building materials for these extracellular structures are polysaccharides that either function on their own or are integrated with other polymers into elaborate composite materials. Mucoid Group A Streptococci produce a thick polysaccharide capsule that consists of hyaluronan (HA). HA is an acidic hetero-polysaccharide primarily produced by vertebrates as an abundant component of the extracellular matrix in soft connective tissues, cartilage, and the vitreous of the eye. Because HA is non- immunogenic, microbial HA capsules are an efficient mechanism to escape complement mediated killing, thereby contributing significantly to streptococcal virulence. Group A streptococcal infections can cause severe illnesses, including rheumatic fever and necrotizing fasciitis. We seek to determine the mechanism by which streptococcal HA capsules are formed. HA is synthesized by a membrane-embedded enzyme (HAS) that performs two tasks. It functions as a (1) glycosyltransferase to synthesize HA from UDP-activated substrates and (2) translocase that secretes HA across the membrane through a channel formed by its own membrane-spanning region. How HAS couples these reactions to secrete an acidic polymer up to ~100,000 sugar units long is currently unknown. The proposed research takes advantage of our detailed biochemical analyses of streptococcal HAS. We demonstrated that the enzyme functions as an obligate dimer in which two protomers form a single HA polymer and likely also a HA channel at their interface. This enzyme complex can be purified and reconstituted into planar membrane bilayers, called nanodiscs, which are excellent membrane surrogates for biochemical and structural analyses. We propose to develop a toolset that will allow us to determine the HAS structure at different states during HA biosynthesis. To this end, under Aim 1 we will generate conformation sensitive Fab antibody fragments that specifically recognize 3-dimensional epitopes of HAS. A primary focus will be on identifying Fab fragments that interact with a single HAS copy in the context of a dimeric assembly, which is expected to facilitate structural analyses by cryo electron microscopy. Further, Fab binders will be selected that recognize and stabilize the HAS dimer interface, which are expected to aid in protein crystallization. In Aim 2, we will generate HAS hyaluronan translocation intermediates to (1) identify the polysaccharide length spanning the enzyme?s transmembrane channel, (2) monitor polymer release from the synthase, and (3) allow structure determination by cryo electron microscopy. Combined, our research will provide a complete toolset necessary to obtain structural snapshots of bacterial hyaluronan biosynthesis along its catalytic cycle.